Medical matters – fungal infections – protocols

These practical protocols are designed for students doing an Extended Project Qualification, Advanced Higher investigation, or IB investigation in biology.

They are linked to our Medical matters – fungal infections – project starter.  

 

Possible areas for investigation
  • Investigate different types of yeast with particular antifungals
  •  Investigate different types of antifungals with one yeast species – could use medical products, veterinary products, herbal product and natural products
  • Compare treatments for thrush with treatments for athletes foot – some products allow growth of the yeast in the inhibition zone after a few days

 

Step-by-step protocols

Starter experiment

  • 1 gram of yeast
  • Bottle of 10 cm3 of sterile distilled water
  • 2 sterile syringes or pipettes
  • sterile spreader
  • number 4 or 5 cork borer
  • sample of anti-fungal treatment
  • 2 glucose yeast agar plates
  • sterile seeker
  • sterile forceps
  • innoculating loops
  • labels
  • ethanol

 

Instructions

  1.  Add one gram of yeast to a bottle of 10 cm3 of sterile distilled water and shake thoroughly.
  2.  Alternatively add a few loopfuls of yeast from a pre-prepared culture to 2 cm3 of sterile distilled water and shake thoroughly.
  3.  Warm this solution to approx 40°
  4.  Pre-label your agar plate with your initial and the date and stating species of yeast and treatment to be used.
  5.  Use a sterile syringe to add 0.3 cm3 of the yeast solution to the agar plate and spread with a sterile spreader. (the spreader can be sterilised by placing in ethanol, flaming and allowing to burn off)
  6.  Flame a 4 or 5 cork borer, use it to make a well in the agar plate and remove thecircle of agar with a sterile seeker.
  7.  Add 0.1 cm3 of anti-fungal treatment to the well
  8.  Seal plate and incubate for 3 days
  9.  Measure the diameter of any area of inhibition over the next 3 to 4 days

 

General equipment list
For a project to investigate fungal inhibition, you are likely to need the following equipment and materials

  • Yeast samples dried, freeze dried or fresh
  • Sterile distilled water
  • Sterile syringes or pipettes
  • Spreader
  • Number 4 or 5 cork borer
  • sample of anti-fungal treatment
  • Glucose yeast agar plates
  • Petri dishes
  • Sterile seeker
  • Sterile forceps
  • Inoculating loops
  • Labels

 

Preparation of Materials
Seekers and forceps can be sterilised by wrapping them in tin-foil and then autoclaving or the students can be taught to alcohol flame them.

Several different yeast preparations can be used:

Saccharomyces cerevisiae can be used taken from a block of yeast.
Phaffia rhodozyma can be grown up on a slope or agar plate.

Both yeasts will grow on Glucose yeast agar ( Phaffia grows at 20°C and Saccharomyces at 35°C.)

Although it would not be possible to use Candida albicans for this investigation Candida utilis is available from National Collection of Yeast Cultures.

Antifungal products can be bought from a chemist shop – Canistan; Daktarin; Diflucan etc. Antifungal Vet products can be bought in pet shops.

 

Making Glucose Yeast Agar – (this method will make enough agar for 3 petri dishes)

  • 100 cm3 beaker
  • stirring rod
  • 3 McCartney bottles
  • weighing boat
  • glucose
  • yeast extract
  • 0.1M Sulphuric Acid
  • distilled water
  • balance (accurate to 0.5g)
  • pH paper
  • 10ml syringe
  • agar
  1. Weigh out 1g of glucose and 0.5g of yeast extract.
  2.  Place the chemicals together in a beaker and add enough distilled water to make up to the 50 cm3 mark on the beaker.
  3.  Stir thoroughly and then test the pH. Add enough sulphuric acid one drop at a time to make the solution pH6 – test with pH probe or pH paper.
  4.  Place 15 cm3 of this solution into a McCartney bottle- you should have enough for 3 bottles.
  5.  Add 0.3g of agar to each McCartney bottle.
  6.  Shake and then place lid loosely on bottle and initial your bottles.
  7.  Autoclave for 15 minutes.

 

 

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